Bottom Line:
However, the cross-talk between TNFα and PKD associated signaling pathways remains elusive.In this study, we found that stimulation of renal epithelial cells with TNFα or RANKL (receptor activator of NF-κB ligand), a member of the TNFα cytokine family, activated either the PI3K pathway, leading to AKT and mTOR mediated the increase of Id2 protein, or MAPK and Cdk2 to induce Id2 nuclear translocation.The effects of TNFα/RANKL on increasing Id2 protein and its nuclear translocation caused significantly decreased mRNA and protein levels of the Cdk inhibitor p21, allowing increased cell proliferation.

Affiliation: Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS 66160, United States of America; Kidney Institute, University of Kansas Medical Center, Kansas City, KS 66160, United States of America.

ABSTRACTTumor necrosis factor alpha (TNFα) is present in cyst fluid and promotes cyst growth in autosomal dominant polycystic kidney disease (ADPKD). However, the cross-talk between TNFα and PKD associated signaling pathways remains elusive. In this study, we found that stimulation of renal epithelial cells with TNFα or RANKL (receptor activator of NF-κB ligand), a member of the TNFα cytokine family, activated either the PI3K pathway, leading to AKT and mTOR mediated the increase of Id2 protein, or MAPK and Cdk2 to induce Id2 nuclear translocation. The effects of TNFα/RANKL on increasing Id2 protein and its nuclear translocation caused significantly decreased mRNA and protein levels of the Cdk inhibitor p21, allowing increased cell proliferation. TNFα levels increase in cystic kidneys in response to macrophage infiltration and thus might contribute to cyst growth and enlargement during the progression of disease. As such, this study elucidates a novel mechanism for TNFα signaling in regulating cystic renal epithelial cell proliferation in ADPKD.

pone.0131043.g008: TNFα and RANKL stimulation regulates the expression of p21 in Pkd1 wild type MEK cells.(A) Western blot analysis of the expression of Id2 and p21 from whole cell lysates of Pkd1 wild type and MEK cells. The numbers at the bottom indicate the relative intensities of the bands, which are normalized to actin. (B and C) TNFα and RANKL stimulation decreased the expression of p21 mRNA (B) and protein (C). n = 3, ANOVA, p < 0.01. The numbers at the bottom indicate the relative intensities of the bands, which are normalized to actin. (D) Western blot analysis of the expression of Id2 and p21 from whole cell lysates of Pkd1 wild type MEK cells treated with p38 activator U-46619 (5 μM). The numbers at the bottom indicate the relative intensities of the bands, which are normalized to actin. (E) Pkd1 wild type MEK cells were transfected with control siRNA or Id2 siRNA for 24 hours, and then treated with TNFα and RANKL for 24 hours. The expression of Id2 and p21 in these cells were analyzed by western blot. The numbers at the bottom indicate the relative intensities of the bands, which are normalized to actin. (F) TNFα and RANKL stimulation increased the proliferation of Pkd1 wild type MEK cells analyzed by MTT assay. n = 4, ANOVA, p < 0.01. (G) TNFα and RANKL mediated pathways in renal epithelial cells.

Mentions:
Our previous studies found that Id2 regulates the cell cycle through downregulation of p21, an inhibitor of CDK2, in cystic renal epithelial cells [18]. Id2 was upregulated, and p21 was downregulated in Pkd1 MEK cells compared with Pkd1 wild type MEK cells (Fig 8A). As TNFα or RANKL increases the expression and nuclear translocation of Id2, next, we examined the expression of p21 in Pkd1 wild type MEK cells upon TNFα or RANKL treatment by qRT-PCR and western blot. We found that treatment with TNFα or RANKL decreased the expression of p21 mRNA (Fig 8B) and protein levels (Fig 8C) in Pkd1 wild type MEK cells. We further found that treatment with an activator of p38, U-46619, increased the expression of Id2 but decreased the expression of p21 in Pkd1 wild type MEK cells (Fig 8D).

pone.0131043.g008: TNFα and RANKL stimulation regulates the expression of p21 in Pkd1 wild type MEK cells.(A) Western blot analysis of the expression of Id2 and p21 from whole cell lysates of Pkd1 wild type and MEK cells. The numbers at the bottom indicate the relative intensities of the bands, which are normalized to actin. (B and C) TNFα and RANKL stimulation decreased the expression of p21 mRNA (B) and protein (C). n = 3, ANOVA, p < 0.01. The numbers at the bottom indicate the relative intensities of the bands, which are normalized to actin. (D) Western blot analysis of the expression of Id2 and p21 from whole cell lysates of Pkd1 wild type MEK cells treated with p38 activator U-46619 (5 μM). The numbers at the bottom indicate the relative intensities of the bands, which are normalized to actin. (E) Pkd1 wild type MEK cells were transfected with control siRNA or Id2 siRNA for 24 hours, and then treated with TNFα and RANKL for 24 hours. The expression of Id2 and p21 in these cells were analyzed by western blot. The numbers at the bottom indicate the relative intensities of the bands, which are normalized to actin. (F) TNFα and RANKL stimulation increased the proliferation of Pkd1 wild type MEK cells analyzed by MTT assay. n = 4, ANOVA, p < 0.01. (G) TNFα and RANKL mediated pathways in renal epithelial cells.

Mentions:
Our previous studies found that Id2 regulates the cell cycle through downregulation of p21, an inhibitor of CDK2, in cystic renal epithelial cells [18]. Id2 was upregulated, and p21 was downregulated in Pkd1 MEK cells compared with Pkd1 wild type MEK cells (Fig 8A). As TNFα or RANKL increases the expression and nuclear translocation of Id2, next, we examined the expression of p21 in Pkd1 wild type MEK cells upon TNFα or RANKL treatment by qRT-PCR and western blot. We found that treatment with TNFα or RANKL decreased the expression of p21 mRNA (Fig 8B) and protein levels (Fig 8C) in Pkd1 wild type MEK cells. We further found that treatment with an activator of p38, U-46619, increased the expression of Id2 but decreased the expression of p21 in Pkd1 wild type MEK cells (Fig 8D).

Bottom Line:
However, the cross-talk between TNFα and PKD associated signaling pathways remains elusive.In this study, we found that stimulation of renal epithelial cells with TNFα or RANKL (receptor activator of NF-κB ligand), a member of the TNFα cytokine family, activated either the PI3K pathway, leading to AKT and mTOR mediated the increase of Id2 protein, or MAPK and Cdk2 to induce Id2 nuclear translocation.The effects of TNFα/RANKL on increasing Id2 protein and its nuclear translocation caused significantly decreased mRNA and protein levels of the Cdk inhibitor p21, allowing increased cell proliferation.

Affiliation:
Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS 66160, United States of America; Kidney Institute, University of Kansas Medical Center, Kansas City, KS 66160, United States of America.

ABSTRACTTumor necrosis factor alpha (TNFα) is present in cyst fluid and promotes cyst growth in autosomal dominant polycystic kidney disease (ADPKD). However, the cross-talk between TNFα and PKD associated signaling pathways remains elusive. In this study, we found that stimulation of renal epithelial cells with TNFα or RANKL (receptor activator of NF-κB ligand), a member of the TNFα cytokine family, activated either the PI3K pathway, leading to AKT and mTOR mediated the increase of Id2 protein, or MAPK and Cdk2 to induce Id2 nuclear translocation. The effects of TNFα/RANKL on increasing Id2 protein and its nuclear translocation caused significantly decreased mRNA and protein levels of the Cdk inhibitor p21, allowing increased cell proliferation. TNFα levels increase in cystic kidneys in response to macrophage infiltration and thus might contribute to cyst growth and enlargement during the progression of disease. As such, this study elucidates a novel mechanism for TNFα signaling in regulating cystic renal epithelial cell proliferation in ADPKD.